Compositions and methods for detection of mycoplasma genitalium

ABSTRACT

Methods for the rapid detection of the presence or absence of Mycoplasma genitalium (MG) in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the target MG gene, along with kits are provided that are designed for the detection of MG.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/601,545, filed May 22, 2017, which claims the benefit of priority ofU.S. Provisional Patent Application Ser. No. 62/342,519, filed May 27,2016, each of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “33594_US1_Sequence_Listing.txt”, having a size in bytesof 30 kb, and created on Apr. 17, 2017. The information contained inthis electronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The present disclosure relates to the field of molecular diagnostics,and more particularly to detection of Mycoplasma genitalium.

BACKGROUND OF THE INVENTION

Mycoplasma genitalium (MG) is a gram-negative bacterium that lacks acell wall and lives on the surface and in the epithelial cells of theurinary and genital tracts of men and women. M. genitalium is a commoncause of nongonococcal urethritis (NGU) in men and an increasinglyrecognized cause of cervicitis and pelvic inflammatory disease (PID) inwomen. A recent meta-analysis has shown that infection with M.genitalium was associated with an approximately two-fold increase inrisk for cervicitis, pelvic inflammatory disease, preterm birth,spontaneous abortion, and infertility in women. The prevalence of M.genitalium in lower risk populations in both men and women has beenreported to be approximately 1% to 3%. In higher risk populations,prevalence of 10% to 41% in men and 7.3% to 14% in women has beenreported. The prevalence of M. genitalium in higher risk populationsoften exceeds that of Neisseria gonorrhoeae and is similar to theprevalence of Chlamydia trachomatis. M. genitalium infections largely gounrecognized, and infected individuals are either asymptomatic or havesymptoms similar to those associated with other bacterial infections ofthe urogenital tract.

The diagnosis of M. genitalium infection has traditionally been cultureof urogenital specimens but culture is insensitive and slow and may takeup to 6 months to recover the organism. Nucleic acid amplification tests(NAATs) for the diagnosis of M. genitalium infection in the UnitedStates and Europe NAATs have been made available as Laboratory DevelopedProcedures (LDPs) and have been shown to have superior sensitivitycompared to culture.

The Center for Disease Control (CDC) recommends that M. genitaliumshould be suspected in cases of persistent or recurrent urethritis andmay be considered in persistent or recurrent cases of cervicitis andpelvic inflammatory disease (PID). The fact that M. genitalium is aslow-growing organism, culture is not readily available, slow andinsensitive, NAATs are the preferred method for M. genitalium detection.In research settings, M. genitalium is diagnosed by NAAT testing ofurine, urethral, vaginal, and cervical swabs and through endometrialbiopsies, typically using in-house PCR or assays intended for researchuse only. Thus there is a need in the art for a quick and reliablemethod to specifically detect MG in a sensitive manner.

SUMMARY OF THE INVENTION

Certain embodiments in the present disclosure relate to methods for therapid detection of the presence or absence of MG in a biological ornon-biological sample, for example, multiplex detection of MG byreal-time polymerase chain reaction in a single test tube. Embodimentsinclude methods of detection of MG comprising performing at least onecycling step, which may include an amplifying step and a hybridizingstep. Furthermore, embodiments include primers, probes, and kits thatare designed for the detection of MG in a single tube. The detectionmethods are designed to target specific genes in the M. genitaliumgenome with a potential to discriminate against the nearest neighbor,Mycoplasma pneumonia, and also other pathogens or commensals found inthe human mouth or urogenital tract.

A method for detecting MG in a sample is provided, including performingan amplifying step including contacting the sample with a set of primersdesigned to target a specific MG gene to produce an amplificationproduct if MG is present in the sample; performing a hybridizing stepincluding contacting the amplification product with one or moredetectable probes to the target MG gene; and detecting the presence orabsence of the amplified product, wherein the presence of the amplifiedproduct is indicative of the presence of MG in the sample and whereinthe absence of the amplified product is indicative of the absence of MGin the sample; wherein the target MG gene is selected from the groupconsisting of the 23s ribosomal RNA (23s) gene, the conserved region Aof the mgpB gene within the MgPa adhesion operon (mgpB), and thevariable EF region of the mgpB partial repeats (MgPar). FIG. 1 shows apicture of the circular M. genitalium genome and locations of the mgpBgene and the nine MgPar partial repeats.

In one aspect a method of detecting Mycoplasma genitalium (MG) in asample is provided, the method comprising performing an amplifying stepcomprising contacting the sample with a set of target MG gene primers toproduce an amplification product if a target MG gene nucleic acid ispresent in the sample; performing a hybridizing step comprisingcontacting the amplification product with one or more detectable targetMG gene probes; and detecting the presence or absence of theamplification product, wherein the presence of the amplification productis indicative of the presence of MG in the sample and wherein theabsence of the amplification product is indicative of the absence of MGin the sample; wherein the set of target MG gene primers comprise afirst primer comprising a first oligonucleotide sequence selected fromthe group consisting of SEQ ID NOs: 1-7, 29-35, and 47-56 or acomplement thereof, and a second primer comprising a secondoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 8-17, 36-41, and 57-66, or a complement thereof; and wherein theone or more detectable target MG gene probes comprises a thirdoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 18-28, 42-46, and 67-89, or the complement thereof.

In one embodiment, the primer set for amplification of the 23s genetarget includes a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 1, 2, 3, 4, 5, 6, and 7, or a complement thereof, and a secondprimer comprising or consisting of a second oligonucleotide sequenceselected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13,14, 15, 16, and 17, or a complement thereof, and the detectable probefor detection of the 23s gene amplification product includes or consistsof the nucleic acid sequences of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24,25, 26, 27, and 28, or a complement thereof.

In certain embodiments, the primer set for amplification of the 23s genetarget includes a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 3 and 4, or a complement thereof, and a second primer comprising orconsisting of a second oligonucleotide sequence selected from the groupconsisting of SEQ ID NOs: 8 and 9, or a complement thereof, and thedetectable probe for detection of the 23s gene amplification productincludes or consists of the nucleic acid sequences of SEQ ID NO: 24, ora complement thereof.

In another embodiment, the primer set for amplification of the mgpB genetarget includes a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 29, 30, 31, 32, 33, 34, and 35, or a complement thereof, and asecond primer comprising or consisting of a second oligonucleotidesequence selected from the group consisting of SEQ ID NOs: 36, 37, 38,39, 40, and 41, or a complement thereof, and the detectable probe fordetection of the mgpB gene amplification product includes or consists ofthe nucleic acid sequences of SEQ ID NOs: 42, 43, 44, 45, and 46, or acomplement thereof.

In certain embodiments, the primer set for amplification of the mgpBgene target includes a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 34 and 35, or a complement thereof, and a second primer comprisingor consisting of a second oligonucleotide sequence selected from thegroup consisting of SEQ ID NOs: 40 and 41, or a complement thereof, andthe detectable probe for detection of the mgpB gene amplificationproduct includes or consists of the nucleic acid sequences of SEQ IDNOs: 45 and 46, or a complement thereof.

In another embodiment, the primer set for amplification of the MgPargene target includes a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 47, 48, 49, 50, 51, 52, 53, 54, 55, and 56, or a complementthereof, and a second primer comprising or consisting of a secondoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 57, 58, 59, 60, 61, 62, 63, 64, 65, and 66, or a complementthereof, and the detectable probe for detection of the MgPar geneamplification product includes or consists of the nucleic acid sequencesof SEQ ID NOs: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, and 89, or a complement thereof.

In certain embodiments, the primer set for amplification of the MgPargene target includes a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 48 and 56, or a complement thereof, and a second primer comprisingor consisting of a second oligonucleotide sequence selected from thegroup consisting of SEQ ID NOs: 65 and 66, or a complement thereof, andthe detectable probe for detection of the MgPar gene amplificationproduct includes or consists of the nucleic acid sequences of SEQ IDNOs: 80 and 89, or a complement thereof.

In some embodiments, the hybridizing step comprises contacting theamplification product with the detectable target MG gene probe that islabeled with a donor fluorescent moiety and a corresponding acceptormoiety; and the detecting step comprises detecting the presence orabsence of fluorescence resonance energy transfer (FRET) between thedonor fluorescent moiety and the acceptor moiety of the probe, whereinthe presence or absence of fluorescence is indicative of the presence orabsence of MG in the sample.

In some embodiments the amplifying and the hybridizing steps arerepeated. Herein, the number of repetitions depends, e.g., on the natureof the sample. If the sample is a complex mixture of nucleic acids, moreamplifying and hybridizing steps will be required to amplify the targetsequence sufficient for detection. In some embodiments, the amplifyingand the hybridizing steps are repeated at least about 20 times, but maybe repeated as many as at least 25, 30, 40, 50, 60, or even 100 times.Further, detecting the presence or absence of the amplification productmay be performed during or after each amplifying and hybridizing step,during or after every other amplifying and hybridizing step, during orafter particular amplifying and hybridizing steps or during or afterparticular amplifying and hybridizing steps, in which—ifpresent—sufficient amplification product for detection is expected. Insome embodiments, the amplifying step employs a polymerase enzyme having5′ to 3′ nuclease activity. In some embodiments, the donor fluorescentmoiety and the corresponding acceptor moiety are within no more than8-20 nucleotides of each other on the probe. In some embodiments, theacceptor moiety is a quencher.

In some embodiments the oligonucleotides comprise or consist of asequence of nucleotides selected from SEQ ID NOs: 1-89, or a complementthereof have 100 or fewer nucleotides, 50 or fewer nucleotides, 40 orfewer nucleotides or 30 or fewer nucleotides. In some embodiments, thefirst and second target MG gene primers and detectable target MG geneprobe have 40 or fewer nucleotides (e.g. 35 or fewer nucleotides, 30 orfewer nucleotides, etc.).

In another embodiment, the present disclosure provides anoligonucleotide that includes a nucleic acid having at least 70%sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) toone of SEQ ID NOs: 1-89, or a complement thereof, which oligonucleotidehas 100 or fewer nucleotides. Generally, these oligonucleotides may beprimer nucleic acids, probe nucleic acids, or the like in theseembodiments. In some embodiments, the oligonucleotides comprise at leastone modified nucleotide, e.g., to alter nucleic acid hybridizationstability relative to unmodified nucleotides. Optionally, theoligonucleotides comprise at least one label and/or at least onequencher moiety. In some embodiments, the oligonucleotides include atleast one conservatively modified variation. “Conservatively modifiedvariations” or, simply, “conservative variations” of a particularnucleic acid sequence refers to those nucleic acids, which encodeidentical or essentially identical amino acid sequences, or, where thenucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. One of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 4%, 2% or 1%) in an encoded sequenceare “conservatively modified variations” where the alterations result inthe deletion of an amino acid, addition of an amino acid, orsubstitution of an amino acid with a chemically similar amino acid. Insome embodiments, at least one of the first and second target MG geneprimers and detectable target MG gene probe comprises at least onemodified nucleotide.

In some embodiments, amplification (the amplifying step) can employ apolymerase enzyme having 5′ to 3′ nuclease activity. Thus, the donorfluorescent moiety and the acceptor moiety, e.g., a quencher, may bewithin no more than 5 to 20 nucleotides (e.g., 8 or 10) of each otheralong the length of the probe. In another aspect, the detectable probeincludes a nucleic acid sequence that permits secondary structureformation. Such secondary structure formation generally results inspatial proximity between the first and second fluorescent moiety.According to this method, the second fluorescent moiety on the probe canbe a quencher.

The present disclosure provides for methods of detecting the presence orabsence of MG in a biological sample from an individual. Such methodsgenerally include performing at least one cycling step, which includesan amplifying step and a dye-binding step. Typically, the amplifyingstep includes contacting the sample with a plurality of pairs of primersdesigned to target a specific MG gene to produce one or more target MGgene amplification products if the target MG gene nucleic acid moleculeis present in the sample, and the dye-binding step includes contactingthe target MG gene amplification product with a double-stranded DNAbinding dye. Such methods also include detecting the presence or absenceof binding of the double-stranded DNA binding dye into the amplificationproduct, wherein the presence of binding is indicative of the presenceof MG in the sample, and wherein the absence of binding is indicative ofthe absence of MG in the sample. A representative double-stranded DNAbinding dye is ethidium bromide. In addition, such methods also caninclude determining the melting temperature between the target MG geneamplification product and the double-stranded DNA binding dye, whereinthe melting temperature confirms the presence or absence of MG. Thetarget MG gene may include but is not limited to the 23s gene, the mgpBgene, and the MgPar partial repeats.

In another aspect, the methods of detecting MG in a biological samplefrom an individual is conducted together with methods to detectTrichomonas vaginalis (TV) from the same biological sample due to theasymptomatic nature of individuals infected with MG and/or TV. Primers,probes and kits used for detecting TV are described in U.S. ProvisionalPatent Application No. 62/342,600, titled “Compositions and methods fordetection of Trichomonas vaginalis”, which is incorporated herein byreference in its entirety. In one embodiment, the methods of detectingMG and TV in the biological sample are performed in the same reactionmixture as a multiplex assay.

In yet another aspect, a kit for detecting one or more nucleic acids ofMG is provided. The kit can include one or more sets of primers specificfor amplification of the target MG gene; and one or more detectableprobes specific for detection of the target MG gene amplificationproducts. The target MG gene may include but is not limited to the 23sgene, the mgpB gene, and the MgPar partial repeats.

In particular, the oligonucleotide primers and probes disclosed above inconnection with the method according to the invention are suitable tobeing included in a kit according to the invention. Herein, a kit fordetecting a nucleic acid of Mycoplasma genitalium (MG) in a sample isprovided comprising a first primer comprising or consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 1-7, 29-35, and 47-56 or a complement thereof; a second primercomprising or consisting of a second oligonucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 8-17, 36-41, and 57-66, or acomplement thereof; and a fluorescently detectably labeled probecomprising or consisting of a third oligonucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 18-28, 42-46, and 67-89, or acomplement, the detectably labeled probe configured to hybridize to anamplicon generated by the first primer and the second primer. In oneaspect, the kit can include probes already labeled with donor andcorresponding acceptor moiety, e.g., another fluorescent moiety or adark quencher, or can include fluorophoric moieties for labeling theprobes. The kit can also include at least one of nucleosidetriphosphates, nucleic acid polymerase, and buffers necessary for thefunction of the nucleic acid polymerase. The kit can also include apackage insert and instructions for using the primers, probes, andfluorophoric moieties to detect the presence or absence of MG in asample. In some embodiments, the third detectably labeledoligonucleotide sequence comprises a donor fluorescent moiety and acorresponding acceptor moiety. In some embodiments, the acceptor moietyis a quencher. In some embodiments, at least one of the first, second,and third oligonucleotides comprises at least one modified nucleotide.In some embodiments, the first, second, and third oligonucleotides have40 or fewer nucleotides.

In another aspect, compositions are provided comprising a set ofoligonucleotide primers for amplifying a target MG gene as disclosedabove. In some embodiments, the set of target MG gene primers comprisesa first primer comprising a first oligonucleotide sequence selected fromthe group consisting of SEQ ID NOs: 1-7, 29-35, and 47-56 or acomplement thereof, and a second primer comprising a secondoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 8-17, 36-41, and 57-66, or a complement thereof. In certainembodiments, the composition further comprises one or more detectabletarget MG gene probes comprising or consisting of a thirdoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 18-28, 42-46, and 67-89, or the complement thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the M. genitalium strain G37genome with the locations of the mgpB gene and the MgPar partial repeatsbeing shown.

FIG. 2 shows PCR growth curves of a real-time PCR experiment in thepresence of various concentrations of genomic M. genitalium DNA template(present in 1000 [black], 100 [light grey] and 10 [grey] genomicequivalent concentrations per PCR reaction).

FIG. 3 shows PCR growth curves of a real-time PCR experiment withconcentrations of genomic M. genitalium DNA template present in 100, 10,5 and 1 genomic equivalent concentrations per PCR reaction (ge/PCR), ina co-amplification with internal control standard and Trichomonasvaginalis DNA template at 10 ge/PCR.

FIG. 4 shows PCR growth curves of the same real-time PCR experiment asshown in FIG. 3 with the amplification of the internal control standardand Trichomonas vaginalis DNA template at 10 ge/PCR.

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis of MG infection by nucleic acid amplification provides amethod for rapidly and accurately detecting the bacterial infection. Areal-time assay for detecting MG in a sample is described herein.Primers and probes for detecting MG are provided, as are articles ofmanufacture or kits containing such primers and probes. The increasedsensitivity of real-time PCR for detection of MG compared to othermethods, as well as the improved features of real-time PCR includingsample containment and real-time detection of the amplified product,make feasible the implementation of this technology for routinediagnosis of MG infections in the clinical laboratory.

The present disclosure includes oligonucleotide primers and fluorescentlabeled hydrolysis probes that hybridize to a specific gene locus of theMG genome in order to specifically identify MG using TagMan®amplification and detection technology. Target selection for MG requireda comprehensive search of the public sequence database, as well asliterature search for MG targets with a potential to discriminateagainst the nearest neighbor, Mycloplasma pneumonia. Targets wereselected for inclusivity purposes as well as for sensitivity purposes.

As a result of the analysis, possible target MG genes include the 23sribosomal RNA gene (GenBank accession number NR077054), the conservedregion A of the mgpB gene within the MgPa adhesion operon (mgpB, GenBankaccession number FJ872586), and the variable EF region of the mgpBpartial repeats (MgPar, GenBank accession number FJ872587). In certainaspects, a dual target approach may be implemented using the wellconserved mgpB gene target. Herein, the well conserved region A of themgpB gene may be selected for inclusivity purposes, and the variable EFregion of the mgpB partial repeats multi-copy target may be selected forsensitivity purposes.

The disclosed methods may include performing at least one cycling stepthat includes amplifying one or more portions of the nucleic acidmolecule gene target from a sample using one or more pairs of primers.“Primer(s)” as used herein refer to oligonucleotide primers thatspecifically anneal to the target gene in MG, and initiate DNA synthesistherefrom under appropriate conditions producing the respectiveamplification products. Each of the discussed primers anneals to atarget within or adjacent to the respective target nucleic acid moleculesuch that at least a portion of each amplification product containsnucleic acid sequence corresponding to the target. The one or moreamplification products are produced provided that one or more of thetarget MG gene nucleic acid is present in the sample, thus the presenceof the one or more of target MG gene amplification products isindicative of the presence of MG in the sample. The amplificationproduct should contain the nucleic acid sequences that are complementaryto one or more detectable probes for target MG gene. “Probe(s)” as usedherein refer to oligonucleotide probes that specifically anneal tonucleic acid sequence encoding the target MG gene. Each cycling stepincludes an amplification step, a hybridization step, and a detectionstep, in which the sample is contacted with the one or more detectableprobes for detection of the presence or absence of MG in the sample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule. Amplifying a nucleicacid molecule typically includes denaturing the template nucleic acid,annealing primers to the template nucleic acid at a temperature that isbelow the melting temperatures of the primers, and enzymaticallyelongating from the primers to generate an amplification product.Amplification typically requires the presence of deoxyribonucleosidetriphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and anappropriate buffer and/or co-factors for optimal activity of thepolymerase enzyme (e.g., MgCl₂ and/or KCl).

The term “primer” as used herein is known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′-OH group whereto further“nucleotides” may be attached by a template-dependent DNA polymeraseestablishing 3′ to 5′ phosphodiester linkage whereby deoxynucleosidetriphosphates are used and whereby pyrophosphate is released. Therefore,there is—except possibly for the intended function—no fundamentaldifference between a “primer”, an “oligonucleotide”, or a “probe”.

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., aC5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an N4-ethyl-dC, an N6-methyl-dA, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides are referredto herein or are otherwise known in the art. In certain embodiments,modified nucleotide substitutions modify melting temperatures (Tm) ofthe oligonucleotides relative to the melting temperatures ofcorresponding unmodified oligonucleotides. To further illustrate,certain modified nucleotide substitutions can reduce non-specificnucleic acid amplification (e.g., minimize primer dimer formation or thelike), increase the yield of an intended target amplicon, and/or thelike in some embodiments. Examples of these types of nucleic acidmodifications are described in, e.g., U.S. Pat. No. 6,001,611, which isincorporated herein by reference.

Detection of MG

The present disclosure provides methods to detect MG by amplifying, forexample, a portion of the target MG gene nucleic acid sequence. Nucleicacid sequences of the 23s ribosomal RNA gene, the mgpB gene and theMgPar partial repeats are publicly available (e.g., GenBank).Specifically, primers and probes to amplify and detect specific MGnucleic acid molecule targets are provided by the embodiments in thepresent disclosure.

For detection of MG, primers and probes to amplify the target MG geneare provided. Nucleic acids other than those exemplified herein can alsobe used to detect MG in a sample. For example, functional variants canbe evaluated for specificity and/or sensitivity by those of skill in theart using routine methods. Representative functional variants caninclude, e.g., one or more deletions, insertions, and/or substitutionsin the target MG gene nucleic acids disclosed herein.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs: 1-89, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs:1-89, or a complement of SEQ ID NOs: 1-89 and the variant.

TABLE I 23s Forward Primers Forward Primers Oligo Name SEQ ID NO:Sequence Modifications JH419BUA 1 GGGGTGGATCACCTCCTTTC<t_BB__dA>t-butylbenzyldA JH407BUC 2 CAATGTTTGGTCTCACAACTAACA<t_BB_dC>t-butylbenzyldC JH409BUA 3 TCCAGTTCTGAAAGAATGTTTTTGA<t_BB_dA>t-butylbenzyldA JH411BUA 4 AAACGACAATCTTTCTAGTTCCAAA<t_BB_dA>t-butylbenzyldA JH413BUC 5 ATGTTTGGTCTCACAACTAACA<t_BB_dC>t-butylbenzyldC JH415BUA 6 CAGTTCTGAAAGAATGTTTTTGA<t_BB_dA>t-butylbenzyldA JH417BUA 7 CGACAATCTTTCTAGTTCCAAA<t_BB_dA>t-butylbenzyldA

TABLE II 23s Reverse Primers Reverse Primers Oligo Name SEQ ID NO:Sequence Modifications JH408BUC  8 CGGATCTCAGGTTTTTACCACCT<t_BB_dC>t-butylbenzyldC JH410BUA  9 CAGATTGCTCCATTCGGAC<_BB_dA> t-butylbenzyldAJH412BUC 10 CAGATTGCTCCATTCGGACA<t_BB_dC> t-butylbenzyldC JH414BUC 11AGATTGCTCCATTCGGACA<t_BB_dC> t-butylbenzyldC JH416BUA 12CACGTCCTTCATCGCCTTTT<t_BB_dA> t-butylbenzyldA JH418BUC 13GATCTCAGGTTTTTACCACCT<t_BB_dC> t-butylbenzyldC JH420BUA 14ATTGCTCCATTCGGAC<t_BB_dA> t-butylbenzyldA JH422BUC 15ATTGCTCCATTCGGACA<t_BB_dC> t-butylbenzyldC JH424BUC 16ATTGCTCCATTCGGACA<t_BB_dC> t-butylbenzyldC JH426BUA 17CGTCCTTCATCGCCTTTT<t_BB_dA> t-butylbenzyldA

TABLE III 23s Probes Probes Oligo Name SEQ ID NO: Sequence ModificationsJH437HQ6 18 <H>GGTCAG<Q>TTTGTATCCAGTT P = phosphate, H = th-HEX,CTGAAAGAATGTTTTTGAAC<P> Q = BHQ2 JH434HQ6 19 <H>GTTCAA<Q>AAACATTCTTTCAP = phosphate, H = th-HEX, GAACTGGATACAAACTGACC<P> Q = BHQ2 JH439HQ6 20<H>GAATGT<Q>TTTTGAACAGTTC P = phosphate, H = th-HEX,TTTCAAAACTGAAAACGACA<P> Q = BHQ2 JH449HQ6 21 <H>TCAGTT<Q>TGTATCCAGTTCTP = phosphate, H = th-HEX, GAAAGAATGTTTTTGAACCAG<P> Q = BHQ2 JH438HQ6 22<H>TGTTCA<Q>AAAACATTCTTTC P = phosphate, H = th-HEX,AGAACTGGATACAAACTGACC<P> Q = BHQ2, JH451HQ6 23 <H>AGAATG<Q>TTTTTGAACAGTTP = phosphate, H = th-HEX, CTTTCAAAACTGAAAACGACA<P> Q = BHQ2 JH441HQ6 24<H>CTAAAA<Q>GGCGATGAAGGAC P = phosphate, H = th-HEX, GTGTTAACCTG<P>Q = BHQ2 JH443HQ6 25 <H>GGTCAG<Q>TTTGTATCCAGTTP = phosphate, H = th-HEX, CTGAAAGAATG<P> Q = BHQ2 JH436HQ6 26<H>GTTCAA<Q>AAACATTCTTTCA P = phosphate, H = th-HEX, GAACTGGATACA<P>Q = BHQ2 JH445HQ6 27 <H>GAATGT<Q>TTTTGAACAGTTCP = phosphate, H = th-HEX, TTTCAAAACTGA<P> Q = BHQ2 JH447HQ6 28<H>CTAAAA<Q>GGCGATGAAGGAC P = phosphate, H = th-HEX, GTGTTAAC<P>Q = BHQ2

TABLE IV mgpB Forward Primers Forward Primers Oligo Name SEQ ID NO:Sequence Modifications JH311BUC 29 GACTTGAAACAATAACAACTTCTCTt-butylbenzyldC T<t_BB_dC> JH313BUA 30 GACTTGAAACAATAACAACTTCTCTt-butylbenzyldA TC<t_BB_dA> JH315BUA 31 AACAATAACAACTTCTCTTCACTAAt-butylbenzyldA AG<t_BB_dA> JH317BUA 32 CAATAACAACTTCTCTTCACTAAAGt-butylbenzyldA ATT<t_BB_dA> JH319BUA 33 ACCCCTTGGACTTGAAACAATAACAt-butylbenzyldA <t_BB_dA> JH321BUA 34 AGAGAACCCAGGATCATTTGG<t_BB_dA>t-butylbenzyldA JH323BUA 35 CTGGAGAGAACCCAGGATC<t_BB_dA> t-butylbenzyldA

TABLE V mgpB Reverse Primers Reverse Primers Oligo Name SEQ ID NO:Sequence Modifications JH310BUA 36 GTTGTTATCATACCTTCTGATTGCAt-butylbenzyldA <t_BB_dA> JH312BUA 37 CTACCGTTGTTATCATACCTTCTG<t_BB_dA>t-butylbenzyldA JH314BUA 38 CATATAAAGCTCTACCGTTGTTATC t-butylbenzyldAAT<t_BB_dA> JH316BUA 39 AATATCATATAAAGCTCTACCGTTG t-butylbenzyldATT<t_BB_dA> JH320BUA 40 TTTTCCATTTTTGCTAAGTTAATAT t-butylbenzyldACATATA<t_BB_dA> JH322BUA 41 GGGGTTTTCCATTTTTGCTAAGTTA t-butylbenzyldA<t_BB_dA>

TABLE VI mgpB Probes Probes Oligo Name SEQ ID NO: Sequence ModificationsJH335HQ6 42 <H>GGAGAG<Q>AACCCAGGATCAT P = phosphate, H = th-HEX,TTGGATTAGTAAGAAGC<P> Q = BHQ2 JH337HQ6 43 <H>AAGATT<Q>ACTGGAGAGAACCP = phosphate, H = th-HEX, CAGGATCATTTGGATTAGTAAG<P> Q = BHQ2 JH339HQ644 <H>CTGGAG<Q>AGAACCCAGGATC P = phosphate, H = th-HEX,ATTTGGATTAGTAAGAAG<P> Q = BHQ2 JH341HQ6 45 <H>CAGCAA<Q>AACTTTGCAATCAP = phosphate, H = th-HEX, GAAGGTATGATAACAACG<P> Q = BHQ2 JH342HQ6 46<H>CGTTGT<Q>TATCATACCTTCT P = phosphate, H = th-HEX,GATTGCAAAGTTTTGCTG<P> Q = BHQ2

TABLE VII MgPar Forward Primers Forward Primers Oligo Name SEQ ID NO:Sequence Modifications JH501BUA 47 TTTCTCCCCTGAATCGGCA<t_BB_dA>t-butylbenzyldA JH503BUA 48 CAACTCCCCCTCCCCTTCA<t_BB_dA> t-butylbenzyldAJH505BUC 49 TCCCCCTCCCCTTCAACTT<t_BB_dC> t-butylbenzyldC JH507BUC 50ATCCCAATTCAGATGATAATAAAGT t-butylbenzyldC CA<t_BB_dC> JH509BUA 51ATCCCAATTCAGATGATAATAAAGT t-butylbenzyldA C<t_BB_dA> JH511BUA 52TCCCACCAGTGACTGGATCA<t_BB_dA> t-butylbenzyldA JLH531 53CAACTCCCACACTGCTTCC<t_BB_dC> t-butylbenzyldC KMMGP560F 54TCCAACTCCCACACTGCTTCCC<t_BB_dC> t-butylbenzyldC KMMGP562F 55TCCAACTCCCACACTGCTTC<t_BB_dC> t-butylbenzyldC KMMGP564F 56CAACTCCCACACTGCTTC<t_BB_dC> t-butylbenzyldC

TABLE VIII MgPar Reverse Primers Reverse Primers Oligo Name SEQ ID NO:Sequence Modifications JH502BUA 57 GGTGAAAAGTTAGGTATAAACACCt-butylbenzyldA C<t_BB_dA> JH504BUA 58 CTGCTCCTGTTCAGATGTC<t_BB_dA>t-butylbenzyldA JH506BUA 59 TGCTCACTATCCTTGTTAAATTG<t_BB_dA>t-butylbenzyldA JH508BUA 60 CACCTCCCCAAACCCAGGTAA<t_BB_dA>t-butylbenzyldA JH510BUA 61 ACCTCCCCAAACCCAGGTAA<t_BB_dA>t-butylbenzyldA JLH536 62 CCTGCTCCCGTTCAGATGT<t_BB_dC> t-butylbenzyldCJLH540 63 CCTGCTCCCGTT<L>AAATGT<t_BB_dC> t-butylbenzyldC,L = 9-(aminoethoxy)- phenoxazine-2′-dC JLH538R 64CCTGCTCCCGTTCA<R>ATGT<t_BB_dC> t-butylbenzyldC R = A or G JLH538R_A 65CCTGCTCCCGTTCAAATGT<t_BB_dC> t-butylbenzyldC JLH538R_G 66CCTGCTCCCGTTCAGATGT<t_BB_dC> t-butylbenzyldC

TABLE IX MgPar Probes Probes Oligo Name SEQ ID NO: SequenceModifications JH521HQ6 67 <H>CTCCCC<Q>ACTTTTTCTAACATP = phosphate, H = th-HEX, CAATGTTGGGGTTAAATCAA<P> Q = BHQ2 JH522HQ6 68<H>TTGATT<Q>TAACCCCAACATT P = phosphate, H = th-HEX,GATGTTAGAAAAAGTGGGGAG<P> Q = BHQ2 JH523HQ6 69 <H>CGCAAT<Q>CAACTGTTGCTCAP = phosphate, H = th-HEX, GAAGCTTACTAGGAA<P> Q = BHQ2 JH524HQ6 70<H>AGTTCC<Q>TAGTAAGCTTCTG P = phosphate, H = th-HEX, AGCAACAGTTGATTGC<P>Q = BHQ2 JLH540HQ6 71 <H>GATTTA<Q>ACCCCAACATTGAP = phosphate, H = th-HEX, TGTTAGAAAAAGTGGGGAG<P> Q = BHQ2 JLH540PDUHQ672 <H>GA<U><U><U>A<Q>ACCCCAAC P = phosphate, H = th-HEX,A<U><U>GA<U>G<U><U>AGAAAA Q = BHQ2, U = propynyldU AG<U>GGGGAG<P>JLH544HQ6 73 <H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGTGGGGAG<P> Q = BHQ2 JLH544PDUHQ6 74<H>A<U><U><U>AA<Q>CCCCAACA P = phosphate, H = th-HEX,<U><U>GA<U>G<U><U>AGAAAAA Q = BHQ2, U = propynyldU G<U>GGGGAG<P>JLH546HQ6 75 <H>GATTTA<Q>ACCCCAACATTGA P = phosphate, H = th-HEX,TGTTAGAAAAAGTGGTGAG<P> Q = BHQ2 JLH546PDUHQ6 76 <H>GATTTA<Q>ACCCCAACAP = phosphate, H = th-HEX, <U><U>GA<U>G<U><U>AGAAAAAG<U>Q = BHQ2, U = propynyldU GGTGAG<P> JLH548HQ6 77<H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGTGGTGAG<P> Q = BHQ2 JLH548PDUHQ6 78<H>ATTTAA<Q>CCCCAACA<U><U> P = phosphate, H = th-HEX,GA<U>G<U><U>AGAAAAAG<U>GG Q = BHQ2, U = propynyldU TGAG<P> JLH550HQ6 79<H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGT<Y>GGGAG<P> Q = BHQ2, Y = 7′deazadG JLH552HQ6 80<H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGTG<Y>GGAG<P> Q = BHQ2, Y = 7′deazadG JLH554HQ6 81<H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGTGG<Y>GAG<P> Q = BHQ2, Y = 7′deazadG JLH556HQ6 82<H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGTGGG<Y>AG<P> Q = BHQ2, Y = 7′deazadG JLH550PDUHQ6 83<H>A<U><U><U>AA<Q>CCCCAACA P = phosphate, H = th-HEX,<U><U>GA<U>G<U><U>AGAAAAA Q = BHQ2, U = propynyldU G<U><Y>GGGAG<P>Y = 7′deazadG JLH552PDUHQ6 84 <H>A<U><U><U>AA<Q>CCCCAACAP = phosphate, H = th-HEX, <U><U>GA<U>G<U><U>AGAAAAAQ = BHQ2, U = propynyldU G<U>G<Y>GGAG<P> Y = 7′deazadG JLH554PDUHQ6 85<H>A<U><U><U>AA<Q>CCCCAACA P = phosphate, H = th-HEX,<U><U>GA<U>G<U><U>AGAAAAA Q = BHQ2, U = propynyldU G<U>GG<Y>GAG<P>Y = 7′deazadG JLH558HQ6 86 <H>ATTTAA<Q>CCCCAACATTGATP = phosphate, H = th-HEX, GTTAGAAAAAGTG<Y>G<P> Q = BHQ2, Y = 7′deazadGKMMGP560HQ6 87 <H>ATTTAA<Q>CCCCAACATTGAT P = phosphate, H = th-HEX,GTTAGAAAAAGTGGG<P> Q = BHQ2 KMMGP562HQ6 88 <H>A<U><U><U>AA<Q>CCCCAACAP = phosphate, H = th-HEX, <U><U>GA<U>G<U><U>AGAAAAAQ = BHQ2, U = propynyldU GTGGG<P> KMMGP564HQ6 89<H>A<U><U><U>AA<Q>C<X>C<X> P = phosphate, H = th-HEX,AA<X>A<U><U>GA<U>G<U><U>AG Q = BHQ2, U = propynyldU AAAAAGTGGG<P>X = propynyldC

In one embodiment, the above described sets of primers and probes areused in order to provide for detection of MG in a biological samplesuspected of containing MG. The sets of primers and probes may compriseor consist of the primers and probes specific for the nucleic acidsequences of the 23s gene, mgpB gene, and the MgPar partial repeatscomprising or consisting of the nucleic acid sequences of SEQ ID NOs:1-89. In another embodiment, the primers and probes for the target MGgenes comprise or consist of a functionally active variant of any of theprimers and probes of SEQ ID NOs: 1-89.

A functionally active variant of any of the primers and/or probes of SEQID NOs: 1-89 may be identified by using the primers and/or probes in thedisclosed methods. A functionally active variant of a primer and/orprobe of any of the SEQ ID NOs: 1-89 pertains to a primer and/or probewhich provides a similar or higher specificity and sensitivity in thedescribed method or kit as compared to the respective sequence of SEQ IDNOs: 1-89.

The variant may, e.g., vary from the sequence of SEQ ID NOs: 1-89 by oneor more nucleotide additions, deletions or substitutions such as one ormore nucleotide additions, deletions or substitutions at the 5′ endand/or the 3′ end of the respective sequence of SEQ ID NOs: 1-89. Asdetailed above, a primer (and/or probe) may be chemically modified,i.e., a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-deazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule encoding the target MGgene, e.g. the 23s gene, can be designed using, for example, a computerprogram such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.).Important features when designing oligonucleotides to be used asamplification primers include, but are not limited to, an appropriatesize amplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 8 to50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength).

In addition to a set of primers, the methods may use one or more probesin order to detect the presence or absence of MG. The term “probe”refers to synthetically or biologically produced nucleic acids (DNA orRNA), which by design or selection, contain specific nucleotidesequences that allow them to hybridize under defined predeterminedstringencies specifically (i.e., preferentially) to “target nucleicacids”, in the present case to a target MG gene nucleic acid. A “probe”can be referred to as a “detection probe” meaning that it detects thetarget nucleic acid.

In some embodiments, the described target MG gene probes can be labeledwith at least one fluorescent label. In one embodiment, the target MGgene probes can be labeled with a donor fluorescent moiety, e.g., afluorescent dye, and a corresponding acceptor moiety, e.g., a quencher.In one embodiment, the probe comprises or consists of a fluorescentmoiety and the nucleic acid sequences comprise or consist of SEQ ID NOs:18-28, 42-46, and 67-89.

Designing oligonucleotides to be used as probes can be performed in amanner similar to the design of primers. Embodiments may use a singleprobe or a pair of probes for detection of the amplification product.Depending on the embodiment, the probe(s) use may comprise at least onelabel and/or at least one quencher moiety. As with the primers, theprobes usually have similar melting temperatures, and the length of eachprobe must be sufficient for sequence-specific hybridization to occurbut not so long that fidelity is reduced during synthesis.Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22,23, 24, or 25) nucleotides in length. In some embodimentsoligonucleotide primers are 40 or fewer nucleotides in length.

Constructs can include vectors each containing one of target MG geneprimers and probes nucleic acid molecules. Constructs can be used, forexample, as control template nucleic acid molecules. Vectors suitablefor use are commercially available and/or produced by recombinantnucleic acid technology methods routine in the art. Target MG genenucleic acid molecules can be obtained, for example, by chemicalsynthesis, direct cloning from MG, or by PCR amplification.

Constructs suitable for use in the methods typically include, inaddition to the target MG gene nucleic acid molecules (e.g., a nucleicacid molecule that contains one or more sequences of SEQ ID NOs: 1-89),sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, and anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

Constructs containing target MG gene nucleic acid molecules can bepropagated in a host cell. As used herein, the term host cell is meantto include prokaryotes and eukaryotes such as yeast, plant and animalcells. Prokaryotic hosts may include E. coli, Salmonella typhimurium,Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts includeyeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cellssuch as COS cells or Chinese hamster ovary (CHO) cells, insect cells,and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. Aconstruct can be introduced into a host cell using any of the techniquescommonly known to those of ordinary skill in the art. For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acids into host cells. Inaddition, naked DNA can be delivered directly to cells (see, e.g., U.S.Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in some embodiments include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin the described target MG gene nucleic acid sequences (e.g., SEQ IDNOs: 1-17, 29-41, and 47-66). A primer can be purified from arestriction digest by conventional methods, or it can be producedsynthetically. The primer is preferably single-stranded for maximumefficiency in amplification, but the primer can be double-stranded.Double-stranded primers are first denatured, i.e., treated to separatethe strands. One method of denaturing double stranded nucleic acids isby heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the described targetMG gene nucleic acid molecules. The temperature for annealing is usuallyfrom about 35° C. to about 65° C. (e.g., about 40° C. to about 60° C.;about 45° C. to about 50° C.). Annealing times can be from about 10 secto about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec toabout 40 sec). The reaction mixture is then adjusted to a temperature atwhich the activity of the polymerase is promoted or optimized, i.e., atemperature sufficient for extension to occur from the annealed primerto generate products complementary to the template nucleic acid. Thetemperature should be sufficient to synthesize an extension product fromeach primer that is annealed to a nucleic acid template, but should notbe so high as to denature an extension product from its complementarytemplate (e.g., the temperature for extension generally ranges fromabout 40° C. to about 80° C. (e.g., about 50° C. to about 70° C.; about60° C.). Extension times can be from about 10 sec to about 5 min (e.g.,about 30 sec to about 4 min; about 1 min to about 3 min; about 1 min 30sec to about 2 min).

PCR assays can employ nucleic acid such as RNA or DNA (cDNA). Thetemplate nucleic acid need not be purified; it may be a minor fractionof a complex mixture, such as nucleic acid contained in human cells.Nucleic acid molecules may be extracted from a biological sample byroutine techniques such as those described in Diagnostic MolecularMicrobiology: Principles and Applications (Persing et al. (eds), 1993,American Society for Microbiology, Washington D.C.). Nucleic acids canbe obtained from any number of sources, such as plasmids, or naturalsources including bacteria, yeast, viruses, organelles, or higherorganisms such as plants or animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target nucleic acid molecules. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, an oligonucleotide probe can contain a donor fluorescentmoiety and a corresponding quencher, which may or not be fluorescent,and which dissipates the transferred energy in a form other than light.When the probe is intact, energy transfer typically occurs between thedonor and acceptor moieties such that fluorescent emission from thedonor fluorescent moiety is quenched the acceptor moiety. During anextension step of a polymerase chain reaction, a probe bound to anamplification product is cleaved by the 5′ to 3′ nuclease activity of,e.g., a Taq Polymerase such that the fluorescent emission of the donorfluorescent moiety is no longer quenched. Exemplary probes for thispurpose are described in, e.g., U.S. Pat. Nos. 5,210,015, 5,994,056, and6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRApair. Commonly used quenchers are DABCYL and TAMRA. Commonly used darkquenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies,Inc., Novato, Calif.), Iowa Black™, (Integrated DNA Tech., Inc.,Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650), (Berry & Assoc.,Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productnucleic acid at the appropriate positions, a FRET signal is generated.Hybridization temperatures can range from about 35° C. to about 65° C.for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a fiber optic light source, orother high intensity light source appropriately filtered for excitationin the desired range.

As used herein with respect to donor and corresponding acceptor moieties“corresponding” refers to an acceptor fluorescent moiety or a darkquencher having an absorbance spectrum that overlaps the emissionspectrum of the donor fluorescent moiety. The wavelength maximum of theemission spectrum of the acceptor fluorescent moiety should be at least100 nm greater than the wavelength maximum of the excitation spectrum ofthe donor fluorescent moiety. Accordingly, efficient non-radiativeenergy transfer can be produced there between.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm can be the distance in Angstroms (Å) from the nucleotide baseto the fluorescent moiety. In general, a linker arm is from about 10 Åto about 25 Å. The linker arm may be of the kind described in WO84/03285. WO 84/03285 also discloses methods for attaching linker armsto a particular nucleotide base, and also for attaching fluorescentmoieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide which contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of MG

The present disclosure provides methods for detecting the presence orabsence of MG in a biological or non-biological sample. Methods providedavoid problems of sample contamination, false negatives, and falsepositives. The methods include performing at least one cycling step thatincludes amplifying a portion of target nucleic acid molecules from asample using one or more pairs of primers, and a FRET detecting step.Multiple cycling steps are performed, preferably in a thermocycler.Methods can be performed using the primers and probes to detect thepresence of MG, and the detection of the target MG gene indicates thepresence of MG in the sample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof MG. TagMan® technology utilizes one single-stranded hybridizationprobe labeled with, e.g., one fluorescent dye and one quencher, whichmay or may not be fluorescent. When a first fluorescent moiety isexcited with light of a suitable wavelength, the absorbed energy istransferred to a second fluorescent moiety or a dark quencher accordingto the principles of FRET. The second moiety is generally a quenchermolecule. During the annealing step of the PCR reaction, the labeledhybridization probe binds to the target DNA (i.e., the amplificationproduct) and is degraded by the 5′ to 3′ nuclease activity of, e.g., theTaq Polymerase during the subsequent elongation phase. As a result, thefluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems) uses TaqMan® technology, and is suitable for performing themethods described herein for detecting the presence or absence of MG inthe sample.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCRmethods. Molecular beacon technology uses a hybridization probe labeledwith a first fluorescent moiety and a second fluorescent moiety. Thesecond fluorescent moiety is generally a quencher, and the fluorescentlabels are typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

Another common format of FRET technology utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in close proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler® Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler®-Red 640 (LC Red 640) or LightCycler® Red 705 (LC Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler® instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of MG genomes). If amplification of targetnucleic acid occurs and an amplification product is produced, the stepof hybridizing results in a detectable signal based upon FRET betweenthe members of the pair of probes.

Generally, the presence of FRET indicates the presence of MG in thesample, and the absence of FRET indicates the absence of MG in thesample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however. Using themethods disclosed herein, detection of FRET within, e.g., 45 cyclingsteps is indicative of a MG infection.

Representative biological samples that can be used in practicing themethods include, but are not limited to respiratory specimens, fecalspecimens, blood specimens, dermal swabs, nasal swabs, wound swabs,blood cultures, skin, and soft tissue infections. Collection and storagemethods of biological samples are known to those of skill in the art.Biological samples can be processed (e.g., by nucleic acid extractionmethods and/or kits known in the art) to release MG nucleic acid or insome cases, the biological sample can be contacted directly with the PCRreaction components and the appropriate oligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the probes from the amplification products canconfirm the presence or absence of MG in the sample.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods. In one embodiment, a LightCycler® instrument isused. The following patent applications describe real-time PCR as usedin the LightCycler® technology: WO 97/46707, WO 97/46714, and WO97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture, compositions or kits to detect MG. An article ofmanufacture can include primers and probes used to detect the target MGgene, together with suitable packaging materials. Compositions caninclude primers used to amplify the target MG gene. In certainembodiments compositions can also comprise probes for detecting thetarget MG gene. Representative primers and probes for detection of MGare capable of hybridizing to target nucleic acid molecules. Inaddition, the kits may also include suitably packaged reagents andmaterials needed for DNA immobilization, hybridization, and detection,such solid supports, buffers, enzymes, and DNA standards. Methods ofdesigning primers and probes are disclosed herein, and representativeexamples of primers and probes that amplify and hybridize to targetnucleic acid molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling theprobes. Examples of suitable FRET donor fluorescent moieties andcorresponding acceptor fluorescent moieties are provided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the primers and probes todetect MG in a sample. Articles of manufacture and compositions mayadditionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes, co-factors, or agents toprevent contamination). Such reagents may be specific for one of thecommercially available instruments described herein.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples, tables and figures are provided to aid theunderstanding of the subject matter, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

Example I

Target selection for MG was the result of a comprehensive search of thepublic sequence database. Multiple methods were used to search forMycoplasma genitalium sequences and its nearest neighbor, Mycoplasmapneumoniae, to design for assay exclusivity. The MG targets selected hadthe most coverage in the public database (mgpB 120 sequences, MgPar 5whole genome assemblies). MG has a small compact genome, which limitedregions to target for NAT design. The mgpB surface protein gene (calledthe MgPar target) was selected for the assay due to its multi-copynumber. Surface proteins are known to undergo recombination, which canresult in a failed assay. To mitigate the associated risks, a dualtarget approach was implemented using a well conserved target (calledthe mgpB target).

The MgPa operon codes three genes: mgpA, mgpB and mgpC (black circlelabeled as mgpB 221 kb in FIG. 1). mgpB (4335 bp) is present in thecomplete operon but there are nine partial repeats (MgPar's) outside ofthe operon. The repeats are partial copies of mgpB (FIG. 1). The wellconserved region A of the mgpB gene (called the mgpB target) wasselected for inclusivity purposes, and the variable region EF (calledthe MgPar target) multi-copy target was selected for sensitivitypurposes (FIG. 1).

Real-time PCR detection of MG were performed using either the Cobas®4800 system or the Cobas® 6800/8800 systems platforms (Roche MolecularSystems, Inc., Pleasanton, Calif.). The final concentrations of theamplification reagents are shown below:

TABLE X PCR Amplification Reagents Master Mix Component Final Conc (50uL) DMSO 0-5.4 % NaN3 0.027-0.030 % Potassium acetate 120.0 mM Glycerol3.0 % Tween 20 0.02 % EDTA 0-43.9 uM Tricine 60.0 mM Aptamer 0.18-0.22uM UNG Enzyme 5.0-10.0 U Z05-SP-PZ Polymerase 30.0-45.0 U dATP400.0-521.70 uM dCTP 400.0-521.70 uM dGTP 400.0-521.70 uM dUTP800.0-1043.40 uM Forward primer oligonucleotides 0.15-0.50 μM Reverseprimer oligonucleotides 0.15-0.50 μM Probe oligonucleotides 0.10 μMManganese Acetate 3.30-3.80 mMThe following table shows the typical thermoprofile used for PCRamplification reaction:

TABLE XVII PCR Thermoprofile Ramp Target Acquisition Hold Rate ProgramName (° C.) Mode (hh:mm:ss) (° C./s) Cycles Analysis Mode Pre-PCR 50None 00:02:00 4.4 1 None 94 None 00:00:05 4.4 55 None 00:02:00 2.2 60None 00:06:00 4.4 65 None 00:04:00 4.4 1st Measurement 95 None 00:00:054.4 5 Quantification 55 Single 00:00:30 2.2 2nd Measurment 91 None00:00:05 4.4 45 Quantification 58 Single 00:00:25 2.2 Cooling 40 None00:02:00 2.2 1 None

The Pre-PCR program comprised initial denaturing and incubation at 55°C., 60° C. and 65° C. for reverse transcription of RNA templates.Incubating at three temperatures combines the advantageous effects thatat lower temperatures slightly mismatched target sequences (such asgenetic variants of an organism) are also transcribed, while at highertemperatures the formation of RNA secondary structures is suppressed,thus leading to a more efficient transcription. PCR cycling was dividedinto two measurements, wherein both measurements apply a one-step setup(combining annealing and extension). The first 5 cycles at 55° C. allowfor an increased inclusivity by pre-amplifying slightly mismatchedtarget sequences, whereas the 45 cycles of the second measurementprovide for an increased specificity by using an annealing/extensiontemperature of 58° C. FIG. 2 depicts a typical amplification experimentwhere PCR growth curves are shown in various concentrations of genomicMG template DNA.

The amplification and detection of the target MG genes, 23s rRNA, mgpB,and MgPar were performed using the conditions described above. Theresults of the experiments using several selected oligonucleotideprimers and probes against genomic MG DNA present at a concentration of1000 genomic equivalent/PCR are shown below as Ct values (thresholdcycle) for the amplification reactions.

TABLE XVIII Amplification and Detection of target MG genes ForwardReverse Ct values Target primer primer Probe 1000 ge/ MG Gene SEQ ID NOSEQ ID NO SEQ ID NO PCR of MG 23s rRNA 3 8 24 28.0 3 9 24 28.1 4 8 2427.3 4 9 24 28.3 mgpB 34 40 45 27.9 35 41 45 27.5 34 40 46 27.2 35 41 4627.5 MgPar 48 66 80 28.2 56 65 80 32.4 48 66 89 28.7 56 65 89 33.1

Example 2

The amplification and detection of the MG mgpB and the MG MgPar geneswas performed as described in Example 1 with the exception that genomictemplate DNA for Trichomonas vaginalis (TV) was included in the PCRassay together with primers and probes that can amplify and detect TV.In this experiment, primers and probes, disclosed in U.S. ProvisionalApplication No. 62/342,600, that hybridize to the 5.8s rRNA gene wereused.

MG Limit of Detection (LOD) was tested at 100, 10, 5 and 1 genomicequivalent concentrations per PCR reaction (ge/PCR), in aco-amplification with internal control standard and TV at 10 ge/PCR. Theresults are shown on FIGS. 3 and 4. All levels of MG were detected withno dropouts, and MG LOD was determined to be <1 ge/PCR.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed:
 1. A method of detecting Mycoplasma genitalium (MG) ina sample, the method comprising: performing an amplifying step by thepolymerase chain reaction (PCR) comprising contacting the sample with aset of target MG gene primers to produce an amplification product if atarget MG gene nucleic acid is present in the sample; performing ahybridizing step comprising contacting the amplification product withone or more detectable target MG gene probes; and detecting the presenceor absence of the amplification product, wherein the presence of theamplification product is indicative of the presence of MG in the sampleand wherein the absence of the amplification product is indicative ofthe absence of MG in the sample; wherein the set of target MG geneprimers and the one or more detectable target MG gene probes amplify anddetect region A of the mgpB gene (mgpB); and wherein the set of targetMG gene primers comprise a first primer consisting of a firstoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 29-35, and a second primer consisting of a second oligonucleotidesequence selected from the group consisting of SEQ ID NOs: 36-41; andwherein the one or more detectable target MG gene probes consists of athird oligonucleotide sequence selected from the group consisting of SEQID NOs: 42-46.
 2. The method of claim 1, wherein: the hybridizing stepcomprises contacting the amplification product with the detectabletarget MG gene probe that is labeled with a donor fluorescent moiety anda corresponding acceptor moiety; and the detecting step comprisesdetecting the presence or absence of fluorescence resonance energytransfer (FRET) between the donor fluorescent moiety and the acceptormoiety of the probe, wherein the presence or absence of fluorescence isindicative of the presence or absence of MG in the sample.
 3. The methodof claim 2, wherein said amplifying step employs a polymerase enzymehaving 5′ to 3′ nuclease activity.
 4. The method of claim 2, wherein thedonor fluorescent moiety and the corresponding acceptor moiety arewithin no more than 8-20 nucleotides of each other on the probe.
 5. Themethod of claim 2, wherein the acceptor moiety is a quencher.
 6. Themethod of claim 1, wherein the first oligonucleotide sequence isselected from the group consisting of SEQ ID NOs: 34-35, the secondoligonucleotide sequence is selected from the group consisting of SEQ IDNOs: 40-41, and the third oligonucleotide sequence is selected from thegroup consisting of SEQ ID NOs: 45-46.
 7. A kit for detecting a nucleicacid of Mycoplasma genitalium (MG) comprising: a first primer consistingof a first oligonucleotide sequence selected from the group consistingof SEQ ID NOs: 29-35, or a complement thereof; a second primerconsisting of a second oligonucleotide sequence selected from the groupconsisting of SEQ ID NOs: 36-41, or a complement thereof; and afluorescently detectably labeled probe comprising a thirdoligonucleotide sequence selected from the group consisting of SEQ IDNOs: 42-46, or a complement, the detectably labeled probe configured tohybridize to an amplicon generated by the first primer and the secondprimer.
 8. The kit of claim 7, wherein the third detectably labeledoligonucleotide sequence comprises a donor fluorescent moiety and acorresponding acceptor moiety.
 9. The kit of claim 8, wherein theacceptor moiety is a quencher.
 10. The kit of claim 7, furthercomprising at least one of nucleoside triphosphates, nucleic acidpolymerase, and buffers necessary for the function of the nucleic acidpolymerase.
 11. The kit of claim 7, wherein at least one of the first,second, and third oligonucleotides comprises at least one modifiednucleotide.
 12. The kit of claim 7, wherein the first oligonucleotidesequence is selected from the group consisting of SEQ ID NOs: 34-35, thesecond oligonucleotide sequence is selected from the group consisting ofSEQ ID NOs: 40-41, and the third oligonucleotide sequence is selectedfrom the group consisting of SEQ ID NOs: 45-46.